Astrocytes are well-known to have almost a full complement of receptors for neurotransmitters. Much of this work, however, has derived from studies on astrocytes in primary cultures, and it remains unclear whether, when or where these receptors are present on astrocytes in situ. The aims of this proposal are to use astrocytes within a few hours of isolation (acute isolation) from different brain regions of postnatal 1 day to older rats, to more directly address these questions. We have been able to prepare acutely isolated preparations in our laboratory, using enzymatic or mechanical dissociation and trituration onto coverslips, and have shown that they can be used to determine the presence or absence of receptor systems linked to ion channels or intracellular Ca2+ increases. The large number of receptors exhibited by primary astrocyte cultures and the key role that receptors and transmitters play in a vast number of neurological disorders, makes it necessary to clearly establish which ones exist on astrocytes in situ and their properties and functions. Studies on astrocytes in slices using conventional or confocal microscopy with electrophysiological or fluorescence techniques are alternative approaches with different advantages and disadvantages. Immunocytochemistry or other visualizing techniques, combined with different levels of microscopic analysis provides important but static data. The acutely isolated cells complements these other approaches. The receptors to be studied will be ATP (P2), adenosine (P1), glutamate and serotonin receptors, and will be studied as a function of the age of the animal and several brain regions. Intracellular changes in (Ca2+) will be studied by microspectrofluorometry, and receptor-linked changes in K+ and C1 channel activity by patch-clamp, single-channel techniques. Up-regulation in culture of receptors not found in the acutely isolated cells will be checked by culturing the cells in serum-containing or serum-free chemically supplemented medium. We have evidence that such up-regulation occurs for both 5-HT2A and P2Y receptors, which are not seen on acutely isolated astrocytes. The problem of false negatives, i.e., receptors not found because of possible damage during isolation, will be approached by immunocytochemistry for the membrane receptor protein, checking that the same cell gives a Ca2+ response to other transmitters, checking for whether cells other than astrocytes in the preparation do show that receptor response, and by use of different enzymes or mechanical dissociation.